Nicotine is well established to be the addictive agent in tobacco, and its metabolites are chemically well described. Surprisingly, the specific enzymes responsible for nicotine metabolism are rather poorly characterized. In this proposal the role of individual P450s in the metabolism of nicotine and cotinine will be investigated. In most smokers the majority of nicotine is metabolized to cotinine. The first step of this pathway is nicotine 5'- oxidation. Cotinine is metabolized to trans-3'-hydroxycotinine, the major nicotine metabolite excreted by smokers. P450 2A6 is reported to be responsible for both the 5'-oxidation of nicotine and the 3'-hydroxylation of cotinine. Nicotine is not exclusively metabolized by P450 2A6, and P450 2A6-catalyzed nicotine metabolism is not specific for 5'-oxidation. We have reported that both P450 2A6 and human liver microsomes (HLM) catalyze the 2'-oxidation of nicotine. The product of this reaction is the precursor of the lung carcinogen, NNK. P450 2A13, which is closely related to P450 2A6, is expressed in the lung and is an efficient catalyst of both nicotine 5'-oxidation and NNK metabolic activation. The extent of nicotine 2'-oxidation by this enzyme is unknown. P450 2A 13 could prove to be an important enzyme in NNK induced lung carcinogenesis in smokers. P450 2B6 is also a catalyst of nicotine 5'-oxidation and appears to contribute to nicotine metabolism by HLM. The antismoking agent bupropion is metabolized by P450 2B6. It is proposed that one mechanism of bupropion's action may be its inhibition of nicotine metabolism. Studies to investigate the importance of nicotine metabolism in smoking and the use of metabolism inhibitors to modify smoking behavior are ongoing. For these studies to succeed and to optimize the use of pharmacological agents to help reduce smoking, it is critical to understand all the enzymes involved in nicotine metabolism and to characterize the products of each pathway. The goals of this proposal are to determine the contribution of P450 2A6 and other hepatic P450s to nicotine and cotinine metabolism, to investigate the metabolism of nicotine and cotinine by human lung microsomes and P450s present in the lung, and to determine the specificity of that metabolism by the important P450s in the lung and liver. The hypotheses to be tested are that both nicotine and cotinine are metabolized by P450s other than P450 2A6 and that the specificity of that metabolism will vary among, P450s. The specific aims are: 1) .to determine the role of human hepatic P450s other than P450 2A6 in both nicotine and cotinine metabolism, 2) to characterize the metabolism of nicotine and cotinine by human lung microsomes, 3) to completely characterize the products of nicotine and cotinine metabolism by human lung microsomes, 4) to determine whether bupropion inhibits any of the pathways of nicotine metabolism in HLM.